ABSTRACT Prosthetic joint infection (PJI), the vast majority of which is caused by Staphylococcal species, is the bane of elective total joint replacement surgery. The pathogenic mechanisms responsible for the unique problems of PJI, which render these infections incurable, remain largely unknown. To address this gap in knowledge, we performed extensive transmission electron microscopy (TEM) studies that uncovered novel, previously unreported mechanisms of S. aureus colonization of canaliculi and submicron cracks in cortical bone. Our novel observations suggest, counter to the well-accepted dogma, that S. aureus must have motility mechanisms that allow it to identify canaliculi and submicron cracks by geometric and rigidity cues from the 3D extracellular matrix of bone, and subsequently deform from spherical cocci into rod shaped bacteria that propel its mitotic progeny through asymmetric septal planes through the submicron canaliculi. This mechanism shelters the S. aureus in these submicron cracks and canaliculi such that leukocytes become incapable of reaching them. This also likely limits the effectiveness of antimicrobials and renders the infection incurable. Thus, our global hypothesis is that S. aureus utilizes haptotaxis and durotaxis (directional mobility guided by geometric and rigidity cues from the 3D extracellular matrix, respectively), to incurably colonize canaliculi and submicron channels in cortical bone. In this application, we take innovative genetic and small molecule screening approaches to design new generations of antimicrobials that inhibit haptotaxis- and durotaxis- mediated colonization of cortical bone. In Aim 1, we use innovative nanoporous silicon membrane transwell chambers to define kinetics of occupancy and migration of S. aureus through submicron channels ex vivo, to simulate in vivo haptotaxis and durotaxis through canaliculi. We also propose to complete a case-control clinical correlate study documenting S. aureus colonization of microcracks and osteocytic-canalicular networks of infected human cortical bone. In Aim 2, we take a focused candidate gene analysis and non-biased de novo genetic screens, with complementary empiric TnSeq mutant library screen approach to identify S. aureus genes involved in canalicular invasion and migration. In Aim 3, we propose to develop 3D-printed spacers infused with novel antibiotics, that target essential enzymes for RNA and protein synthesis in biofilm- associated bacteria or essential proteins involved in haptotaxis and durotaxis, to demonstrate the efficacy of single-stage revision of septic femoral plates in an established OM murine model. The multidisciplinary approach, encompassing tissue engineering and 3D printing, microbial genomics, and high throughput screening of small molecule antimicrobials, will provide critical information needed to solve the significant clinical problems of bone infection by formally understanding this process, identifying novel drug targets, and exploring the potential of localized delivery using 3D-printed antibiotic-impregnated spacers for single-stage revision surgery.